EFFECT OF LACTOBACILLUS ACIDOPHILUS ON SHELF-LIFE OF LOW SALT SOFT CHEESE

Dept. of Food Hygiene,

Fac. of Vet. Med. Kafrelsheikh Univ., Egypt.

Effect Of Lactobacillus Acidophilus On Shelf-Life Of Low Salt SoftCheese

(With 4 Tables)

By

Azza M.K. Sobeih; Ekbal M.A. Ibrahim* and

Hend A. Elbarbary*

* Dept. of Food Control, Fac. of Vet. Med. Benha Univ., Egypt.

(Received at 19/3/2011)

تأثيـر اللاکتوبسلس اسيدوفيلس على مدة صلاحية الجبن الطري

منخفض الملح

عزة محمود کامل صبيح ، إقبال محمد عادل إبراهيم ،

هند أحمد البربري

يعتبر اللاکتوبسلس اسيدوفيلس من البادئات الحيوية صديقة للإنسان حيث يستوطن الأمعاء ويمنع الإصابة بالعديد من الأمراض بالإضافة إلى تأثيره على منتجات الألبان کمادة حافظة طبيعية. أجرى هذا البحث لتصنيع جبن طرى منخفض الملح (3%NaCl) وإضافةاللاکتوبسلس اسيدوفيلسبترکيزي (1٪ و3٪Lb) ودراسة الخواص الحسية ، الکيميائية والميکروبيولوجية للجبن الناتج أثناء فترة الحفظ في الثلاجة. تم اخذ عينات في أول يوم بعد التصنيع ثم على فترات متساوية کل ثلاث أيام حتى فساد الجبن ظاهرياَ. وجد أن الجبن المضاف إليه 3٪Lb أفضل من حيث التقييم الحسي واحتفظ بخواص حسية جيدة حتى اليوم 24 من الحفظ بالثلاجة بينما فسدت الجبن المضاف إليه 1٪Lb بعد 18 يوم والجبن الغير معامل  بعد 15 يوم. ولقد تناقصت قيمة pH وتزايدت نسبة الحموضة في جميع العينات أثناء فترة الحفظ. وقد أوضحت النتائج أن العدد الکلى لبکتريا القولون والفطريات والخمائر اقل  في الجبن المضاف إليه Lb عن الغير معامل، واستمرت هذه الميکروبات في التزايد في الجبن الغير معامل حتى نهاية الصلاحية حيث وصلت إلى  5‚7x10 ⁴ , 14‚8x10 ³ , 21‚4x10 ³ خلية/ جم على التوالي. بينما کانت الزيادة فى الجبن المضاف إليه Lb  في بداية فترة الحفظ ثم انخفضت تدريجياَ. وسجلت1٪Lb الأعداد 93‚x10 , 12‚9x10 ²و 25‚5x10 ² خلية/ جم (في اليوم 18) بينما سجلت 3٪Lb 3‚2x10 , 1‚1x10 ² و 96‚6x10 ²خلية/ جم( في اليوم 24) لکل من بکتريا القولون, الفطريات والخمائر على التوالي. دلت النتائج على خلو جميع العينات من ميکروب الايشريشا کولاى خلال فترة الحفظ وهو ما يتفق مع المواصفة القياسية المصرية. وبالنظر إلى اللاکتوبسلس اسيدوفيلس فقد استطاع البقاء حياَ في کلتا المعاملتين وتزايد العدد تدريجياَ وباستمرار حتى نهاية الصلاحية ووصل إلى 34‚1x10 ⁸ و18‚2x10 ⁹ في 1, 3٪Lb. ويستنتج من الدراسة أن إضافة 3٪ لاکتوبسلس اسيدوفيلس في تصنيع جبن طرى منخفض الملح (3٪ (أفضل حيث تزايد العدد إلى أعلى من الموصى به للحصول على الفوائد الصحية وکذلک امتدت فترة حفظ الجبن بالثلاجة عن الجبن الغير معامل وکذلک الجبن المعامل بنسبة 1٪Lb.

Summary

Lactobacillus acidophilus is one of the most commonly used probiotics. This study aimed to produce low salt (3% Nacl) soft cheese with acceptable organoleptic characters and prolonged shelf-life using Lb.acidophilus (1 and 3%) as bio preservative. The obtained cheese (control, 1%Lb. and 3%Lb.) were kept in refrigerator, sampled fresh (zero time) and at 3days intervals till signs of spoilage were detected. Samples were examined organoleptically, chemically and microbiologically. Results showed that 3% Lb. cheese was superior to 1%Lb. and control cheese when fresh with an average organoleptic overall score of 95.96, 94.04 and 90.82, respectively. pH values at zero time for control, 1% Lb. and 3% Lb. cheese were 6.42, 6.33 and 6.17 then decreased at the end of shelf life (at 15^th, 18^th and 24^th day)to 5.89, 5.65 and 5.43, respectively. Average coliforms count (MPN/g) was 3.6x10¹, 1.1x10¹ and 0.93x10¹ at zero time then reached 7.5x10⁴, 0.93x10¹ and 2.3x10¹ at the end of shelf life for control, 1% Lb. and 3% Lb. cheese, respectively. While, E.coli was absent from all examined low salt soft cheese throughout the entire period.On storage, Lb.acidophilus was -sharply increased in their numbers-1.34x10⁸ and 2.18x10⁹ cfu/g for 1% Lb. (at the 18^th day) and 3% Lb. cheese (at the 24^th day). Effect of Lb. acidophilus strain on mould and yeast count were highly significant (P<0.01). In conclusion, low salt soft cheese with 3% Lb.acidophilus culture had better organoleptic score, microbiological quality and prolonged shelf-life than control and 1% Lb cheese.

Key words: Lactobacillus acidophilus, low salt, soft cheese, shelf-life.

Introduction

Lactobacillus acidophilus is known as probiotics or friendly bacteria (Ljungh and Wadstrom, 2006). Probiotics are mono or mixed culture of viable, defined micro-organisms with sufficient numbers that beneficially affect the host health through altering the intestinal microflora by implantation or colonization (Fuller, 1994; Schrezenmeir and de VrEse, 2001). The probiotic culture of lactic acid bacteria (LAB) had antagonistic actions against many intestinal and food borne pathogens. Different mechanisms of action such as organic acid, bacteriocins and others seem to be involved in this antibacterial activity (Millette et al., 2007)

Lactobacillus acidophilus occurs naturally in the human and animal gastrointestinal tract and has the ability to implant in the intestine and restore the normal intestinal flora, ferments lactose into lactic acid which responsible for low pH and more acidic media which attributed to its therapeutic role in prevention and treatment of many intestinal diseases (Gilliland, 1979; Sandine, 1979; Kandler andWeiss 1986).

Many health benefits have been documented for use of certain Lb. acidophilus strains as a dietary adjunct including; pathogens interference, immune stimulant, alleviation the symptoms of lactose intolerance people, reduction of serum cholesterol level and blood pressure also decrease incidence and duration of diarrhea and common infectious diseases as rhinopharyngitis (Montes et al., 1995; Anderson and Gilliland, 1999; Chou and Weimer, 1999; Parodi, 1999; Guillemard et al., 2010). Moreover, it was recorded that the much lower incidence of colon cancer in northern people was associated with significant and periodical consumption of fermented foods containing probiotics (Lidbeck et al., 1991; Mc Intosh, 1996). Hence, the concept of functional food has known as food or food ingredient with positive effect on host health beyond its nutritive value (Huggett and Verschuren, 1996).

Therapeutically, Lb. acidophilus is considered the most potential probiotics (Klantschitsh et al., 1996) and there is increasing evidence that the regular consumption of foods containing specific strains of lactobacilli as probiotic cultures has beneficial effect on the functioning of the human intestine (Fooks et al., 1999; Mattila-Sandholm et al., 1999; Ouwehand et al., 1999). The most popular vehicle for incorporation of Lb. acidophilus into diet is fermented milk products as soft cheese.

Although sodium chloride is an important ingredient for cheese manufacture which exerts a major influence on its composition, microflora, ripening, texture, flavor and quality (Salem and AbeId, 1997), but high sodium chloride intake has been claimed to be a major contributor to development of hypertension and cardiovascular diseases, therefore low levels of sodium chloride intake is highly recommended for all consumers (El-Abd et al., 2003; Drake et al., 2011). As well as, high salt content used can limit growth of starter organisms and that other salt tolerant flora may be responsible for the developed acidity.

Manufacture of soft cheese by using reconstituted dried skim milk is aiming to improve body and texture character and nutritional values of cheese by raising its total solid content (Abou-Donia, 1991; El-Sheikh   et al., 2001). Furthermore, the protein content of cheese increased by lowering its fat content and as a result cheese becomes of high nutritive value (Chen et al., 1991 and Zommar, 2000). Moreover, Lb. acidophilus DSMZ 2552 can grow well in skim milk at pH up to 4.37 (Metwally     et al., 2005).

This study aimed to produce low salt soft cheese (3% NaCl) with acceptable organoleptic characters and prolonged shelf-life by using Lb. acidophilus as bio preservative and assessment of organoleptic, chemical and microbiological characteristics of manufactured cheese.

Materials and Methods

1. Culture activation:

Lyophilized single strain culture of Lactobacillus acidophilus (Lb.) DSMZ 20079 was obtained from Cairo-MIRCEN, Faculty of Agriculture, Ain-ShamsUniversity, Cairo, Egypt.

The Lyophilized culture of Lb. acidophilus was firstly propagated into MRS broth and incubated at 37°C for 24h for three successive transfers. Then the strain was sub cultured into reconstituted sterile skim-milk powder and incubated at 37°C for 24h in order to further activate the bacterial strain and increase its number to the suitable probiotic dose (10⁷cfu/g). This Lb. acidophilus culture was inoculated during the manufacture of soft cheese.

2. Cheese manufacture:

Low salt soft cheese was manufactured as described by Mehanna and Rashed (1990); El-Sheikh et al. (2001) with slight modification. Reconstituted skim milk powder (<1.25% fat,< 32% protein and >53% lactose) was used for manufacture of cheese with 3% NaCl.

The bulk volume was divided into 3 groups, the first was regarded as control, the second and third were inoculated by 1% Lb. acidophilus culture (1% Lb.) and 3% Lb. acidophilus culture (3% Lb.). The three groups were kept at 42°C till proper curd was obtained, then the curd was kept to drain for 18h in a previously sterilized stainless steel frames lined with cheese cloth.

The obtained cheese with their respective whey were packaged in pre-sterilized aluminum cups and tightly covered with aluminum foil paper then kept at refrigerator. Cheeses were sampled fresh (zero time) and at equal intervals of 3 days till the sings of spoilage were detected.

The experiment was repeated in triplicates and average results for each group (control, 1% Lb. and 3% Lb. cheese) were recorded.

3. Cheese analysis:

3.1. Organoleptic evaluation:

Cheese samples were examined for appearance (10 points), body and texture (60 points) and for flavor (30 points) and the overall score was100 points according to Bodyfeltet al. (1988).

3.2. Chemical examination

Cheese samples were examined for titratable acidity (T.A%) and pH according to Pearson (1984).

3.3. Microbiological examination

Cheese samples were homogenized with sodium citrate (2%) and tenth fold serial dilutions were prepared as described by BSI (1984). The prepared samples were examined for total coliforms count "MPN" (APHA, 1992); Lactobacilli count (Dave and Shah, 1996); E.coli count (APHA, 1992) as well as mould and yeast count (Koburger and Farahat, 1975).

4. Statistical analysis:

Data obtained were statistically analyzed by ANOVA test according to Clarke and Kempson, (1997).

Results

Table 1: Influence of Lb. acidophilus on organoleptic characteristics of low salt soft cheese

* = Significant differences (P< 0.05)          ** = High significant differences (P< 0.01)          S = spoiled

Table 2: Influence of Lb. acidophilus on acidity of the examined samples of low salt soft cheese

 * = Significant differences (P< 0.05)    ** = High significant differences (P< 0.01)  S = spoiled

Table 3: Influence of Lb. acidophilus on bacteriological aspect of the examined samples of low salt soft cheese

  ** = High significant differences (P< 0.01)                            S= spoiled

Table 4: Influence of Lb. acidophilus on mycological aspect of the examined samples of low salt soft cheese

   ** = High significant differences (P< 0.01)                                         S= spoiled

Discussion

Table 1 showed the organoleptic evaluation of the manufactured cheese samples. In general, cheese inoculated with 3% Lb. acidophilus was superior to 1% Lb. and control cheese samples when fresh with an average organoleptic overall score of 95.96, 94.04 and 90.82, respectively. As well as, this superiority continued till 24 days of refrigerated storage with an average overall score of 78.87 for 3% Lb. cheese (Table 1). Such variation was significant at p<0.05. Nearly similar scores were recorded by El-Shibiny et al. (2005). While higher organoleptic scores were recorded by El-Zayat and Osman (2001).

Addition of LAB starter culture was recorded to improve the quality as well as the organoleptic characteristic of fresh, soft and Domiati cheese (Effat, 2000; Mehanna et al., 2002; Shin et al., 2004; Dpesic and JOvanovic, 2005; Dabiza, 2008).

El-Shibiny et al. (2005) found that probiotic soft cheese with 2% salt was superior than control cheese and this continued till the 20^th day of storage. As well as, AbdAlla et al. (2008) stated that probiotic Ras cheese get higher score than traditional control Ras cheese.

Concerning chemical indices, pH values of the examined low salt soft cheese samples (control, 1% Lb. and 3% Lb.) were 6.42, 6.33 and 6.17, respectively at zero time and decreased to 5.89, 5.65 and 5.43 at the end of shelf-life (at 15^th, 18^th and 24^th days of storage), respectively (Table 2).

Lower pH values were recorded by El-Zayat and Osman (2001); El-Abd et al. (2003) and El-Shibiny et al. (2005).

The relatively high pH values at zero time of cheese manufacture may be attributed to the time of drainage as the retention of calcium phosphate increased within the curd matrix, which act as a buffering agent against the developed acidity of cheese (Johnson et al., 1998).

Low salted cheese (3% and 5%) with added mesophilic starter showed higher acidity than control without starter cheeses either when fresh or throughout storage for 60 days and this is due to the action of starter culture (Kehagias et al., 1995 and El-Abd et al., 2003). The increase in titratable acidity controlled the rate of bacterial growth as it acts as bactericidal agent (El-Abd et al., 2003).

As well as, results in Table 2 revealed that cheese samples with lactobacillus acidophilus culture showed slightly higher acidity than the control ones. These results agree with El-ShibinY et al. (2005) and Dabiza (2008). On the day of manufacture the average T.A% were 0.25, 0.27 and 0.28 for control, 1% Lb and 3% Lb. cheese, respectively (Table 2).

During storage, the T.A% of all cheese samples were increased as the storage period progressed, while the pH values showed an opposite trend. These results agreed with those recorded by El-Sissi (1996) and El-Abd et al. (2003). After, the 15^th, 18^th and 24^th day of refrigerated storage, the T.A% reach 0.34, 0.42 and 0.51 for control, 1% Lb. and 3% Lb. cheese, respectively (Table 2). Higher results were recorded by El-Zayat and Osman (2001) and El-Shibiny et al. (2005). It was recorded that T.A% of cheese was greatly affected by salt level and the level of starter culture (El-Abd et al., 2003).

Regarding coliforms, Table 3 showed the effect of using Lb. acidophilus in manufacture of low salt soft cheese on coliforms count. The mean coliforms count was 3.6x10¹, 1.1x10¹ and 0.93x10¹ MPN/g at zero time then reached to 7.5x10⁴, 0.93x10¹ and 2.3x10¹ MPN/g at the 15^th, 18^th and 24^th of refrigerated storage for control, 1% Lb. and 3% Lb. cheese, respectively. In spite of coliforms count in 3% Lb. cheese were higher than the EOSQ (2005) that stipulate less than 10 cfu/gm but it is much lower than counts in control cheese.

Rheem et al. (2002) and El-Abd et al. (2003) recorded that low coliforms count in low salt Domiati cheese is possibly due to the high acidity and production of other antimicrobial substances by action of LAB culture. Furthermore, the preserving effect of LAB are due to production of wide range of antimicrobial metabolites as organic acids, diacetyl, hydrogen peroxide and bacteriocin which have the advantage in competition with other microorganisms including pathogens and other harmful (Oyetayo et al., 2003; marteinez Bveno et al., 2007)

The extended shelf-life of low salt soft cheese with 3% Lb. acidophilus up to the 24^th day of refrigerated storage with restricted and relatively low coliforms may be due to the suppressive effect of several antimicrobial metabolites produced by the added Lb. acidophilus on coliforms.

On the other hand, E.coli were absent from all examined low salt soft cheese (control, 1% and 3% Lb. cheese samples) throughout the entire period (Table 3). This result came in accordance with EOSQ (2005) for cold stored soft cheese, that it must be free from E.coli.

The antimicrobial activity of lactobacilli is associated with the production and synergistic activity of organic acids and hydrogen peroxide, whereas their antagonistic activity against gram-negative and gram-positive bacteria is dependent on the fermentation group of lactobacilli (Annuk et al., 2003).

Table 3 revealed that the mean values of lactobacilli count at zero time were 6.72x10⁶ and 2.43x10⁷ cfu/g for 1% Lb. and 3% Lb. cheese, respectively. On storage, Lb. acidophilus were sharply increased in their numbers and reached 1.34x10⁸ cfu/g for 1% Lb. cheese at the 18^th day and 2.18x10⁹ cfu/g for 3% Lb. cheese at the 24^th day of refrigerated storage (Table 3).

This viable Lb. acidophilus count met the requirements for successful probiotic functional foods that should contain at least 10⁷ cfu/g or ml at the time of consumption to promote their healthy benefits (IDF, 1988; Ishibashi and Shimamura, 1993, El-Shibiny et al., 2005 and Marcatti et al., 2009).

The extend shelf-life of 3% Lb. cheese is probably due to rapid development of titratable acidity in cheese manufactured with added Lb. acidophilus starter culture, compared with less acid development in control cheese samples. Survival of Lb. acidophilus could be attributed to its ability to grow at low pH as acid tolerant organisms. These results are nearly agreed with El-Zayat and Osman (2001) and El-Abd et al. (2003). The degree of survival and activity of Lb. acidophilus depend on salt content and the level of acidity in soft cheese (Mehanna et al., 2002).

Yeasts and moulds play an important role in the spoilage of dairy products, primarily in fermented milks and cheese (Jakobsen and Narvhus, 1996; Welthagen and Viljoen, 1998). Low pH of fermented milk caused by the growth of LAB renders such foods as a good medium for the highly opportunistic fungi to proliferate and thrive leading to spoilage of such products (Batish et al., 1993).

Table 4 declared that, at zero time the average mould count in all examined low salt soft cheese samples were <10 cfu/g and average yeast count for control, 1% Lb. and 3% cheese were 2.9x10¹ ,6.3x10¹ and 1.5x10¹ cfu/g. The effect of Lb. acidophilus strain on mould and yeast count were highly significant (at P<0.01), as their growth were restricted in 3% Lb. and 1% Lb. compared with control cheese. This is may be due to inhibitory effect of antifungal metabolites produced by Lb. acidophilus (Cassandra et al., 2004)

Both mould and yeast were detected in acidophilus cheese after 18 days for 1% Lb. cheese & up to 24 days for 3% Lb. cheese with an average count (cfu/g) of 9.12x10² & 1.1x10² for mould and 3.25x10² & 6.96x10² for yeast. These results exceed the permissible limit (<10 cfu/g for mould and <400 cuf/g for yeast) suggested by Eosq (2005).

Many moulds find cheese an excellent medium for their growth and the cheese become undesirable with musty off-flavors (Abu Sree, 1997). Whiletypical defects caused by yeasts are gas production, discoloration, change in the texture and yeasty flavors (Tudor and Board, 1993). Moreover, the potentially toxigenic species within the genera Penicillium, Aspergillus and Fusarium were detected in cheeses by Montagna et al. (2004).

In conclusion, low salt soft cheese (3% Nacl) with added Lactobacillus acidophilus culture at concentration of 3% had better organoleptic score, micrbiological quality and prolonged shelf-life (24 days) at refrigerated storage.

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